Aggregation of the RNA binding protein TDP-43 (TAR DNA-binding protein 43) is a common pathological hallmark shared by several age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The predominantly nuclear TDP-43 normally undergoes liquid-liquid phase separation (LLPS), in which a homogeneous solution separates into two compartments resembling oil droplets in vinegar.

Intranuclear TDP-43 can phase-separate under physiological conditions. Cellular stress can induce cytoplasmic TDP-43 liquid droplets, which can transition to a solid state, suggesting that TDP-43 aggregation observed in neurodegeneration could be initiated by LLPS. The mechanisms that drive phase separation and aggregation remain unclear.

We identified key regulatory mechanisms of TDP-43 phase separation in cultured cells and in neurons of the rodent nervous system. RNA binding–deficient TDP-43, produced by ALS- or FTD-causing mutations or posttranslational acetylation in its RNA recognition motifs, phase-separated into anisosomes (i.e., droplets with symmetrical liquid spherical shells and liquid cores). RNA-free TDP-43 was found to be enriched in anisosomal shells at concentrations 50 times those of the surrounding nucleoplasm. Acetylation promoted anisosomal formation by abolishing RNA interaction with TDP-43. Anisosomal shells exhibited birefringence (i.e., evidence of a liquid crystal compartment formed from proteins within living cells). Shells were densely packed, as determined with cryo–electron tomography, producing a membraneless, selective barrier to some nuclear proteins and RNAs.

Our mathematical modeling predicted that anisosomes were driven by a core component that self-interacted, weakly bound TDP-43, and did not bind RNA. Guided by these concepts, we used proximity labeling and quantitative proteomics to identify HSP70 chaperones as the primary anisosomal core components. HSP70 chaperones selectively bound to and stabilized RNA-unbound TDP-43. Inhibiting adenosine triphosphate (ATP)–dependent chaperone activity of the HSP70 family or reducing cellular ATP levels induced rapid conversion of TDP-43 anisosomes into uniform gels. Transient proteasome inhibition, mimicking the known reduction in proteasome activity during aging, provoked TDP-43 demixing into anisosomes in neurons in rodents. Postmortem ATP reduction was sufficient to convert anisosomes into aggregates similar to those found in neurodegenerative disease.

We identified how phase separation of the RNA-binding protein TDP-43 can be regulated through RNA binding, disease-causing mutation, posttranslational modification, or chaperone activity inside cells. RNA binding–deficient TDP-43 demixed into anisosomes with cores that could be characterized as a “liquid inside a liquid inside a liquid.” Chaperone activity of the HSP70 family was required to maintain liquidity of anisosomal shells and cores. When ATP levels fell, anisosomes converted into protein aggregates, consistent with being precursors of the pathological aggregates found in patient brain tissues. These findings suggest an essential partnership between TDP-43 and HSP70 chaperones in driving RNA-unbound TDP-43 phase separation into anisosomes and preventing TDP-43 aggregation.

RNA binding protein TDP-43 forms aggregates in degenerating neurons, a pathological feature associated with aging, genetic, and/or environmental factors. Although naturally …